WO2016110864A1

WO2016110864A1 - Process for purification of artemisinin and other constituents from artemisia annua in high yield and high purity

Info

Publication number: WO2016110864A1
Application number: PCT/IN2015/000133
Authority: WO
Inventors: Ashish Ompakash MANTRI
Original assignee: Mantri Ashish Ompakash
Priority date: 2015-01-05
Filing date: 2015-03-19
Publication date: 2016-07-14

Abstract

The present invention discloses a novel hybrid process for the extraction and purification of artemisinin and other constituents from Artemisia annua. The process involves the solid- liquid extraction and chromatographic purification coupled with refining by bleacher followed by evaporative crystallisation to give the artemisinin with yield of >90% and HPLC assay purity of >99%.

Description

Title

Process for purification of artemisinin and other constituents from Artemisia annua in high yield and high purity

Field of the invention

The present invention relates to a process of extraction and purification of Artemisinin from Artemisia to get with yield of >90% and HPLC assay purity of >99%. More particularly the present invention discloses a process of extraction by organic solvent and preparative scale chromatographic purification followed by evaporative crystallisation to obtain artemisinin in high yield and high purity.

Background of the invention

Malaria is a life threatening disease caused by Plasmodium parasites that are transmitted to people through bites of infected mosquitoes. In 2010, 210 million people were affected with malaria causing fatalities of 6.6 million people. Plasmodium falciparum and Plasmodium vivax are the most common parasites causing malaria. Plasmodium falciparum is the most deadly parasite. The artemisinin based combination therapy (ACT) is the best treatment available till date for the treatment of falciparum malaria. The previous generation medicines like chloroquine and sulphadoxine-pyrimethamine (SP) become ineffective as the parasites have developed resistance to them. The artemisinin is the main component of the ACT. The derivatives arteether and artesunate of artemisinin have been widely used for the treatment of malaria.

Artemisinin is a sesquiterpene lactone endoperoxide present in the aerial parts of herb Artemisia annua. The artemisinin is the drug of choice by World Health Organisation for the treatment of malaria therapy. The Artemisia annua is an Asian herb found in China, now cultivated in major parts of Africa and also naturalised in India. The artemisinin is mainly obtained from the leaves of this medicinal herb Artemisia annua L. The artemisinin is also synthesised by chemical route but yields are not promising. The artemisinin can also be produced semi-synthetically. The genetically modified organisms are used for the production of artemisinin precursor by using fermentation. The precursor artemisinic acid is then converted chemically to artemisinin. The chemical conversion of artemisinic acid into artemisinin is critical step and involves photo-oxidation. The yield of this step is not significant. The synthetic and semi-synthetic route of the artemisinin production is not feasible at large scale as it is not economic owing to its low yield. The artemisinin content in the Artemisia annua leaves on dry weight basis varies from 0.01 to 1.4%. All the commercial processes till today produce artemisinin from the herb Artemisia annua. The process of developed by Sanofi Aventis for the production of artemisinic acid precursor to artemisinin lacks economic viability.

The isolation method reported by Hala N. Elsohly involves the liquid-liquid extraction and silica column chromatography to yield the 0.12% w/w (raw material weight) artemisinin with purity of >99%. This method involves the use of multiple solvents and productivity with respect to total solvent consumption and time was low. The supercritical fluid extraction method reported by Tzeg et. al. used supercritical carbon dioxide for extraction whereas purification was performed on silica column chromatography using mixture of ethyl acetate and hexane. The yield of this method is around 54% with purity of artemisinin around 98%. This method requires the setup for the supercritical fluid handling. One of the methods developed by J. Xu et al uses ultrasound assisted extraction using ethanol as a solvent for extraction followed by charcoal column chromatography in stepwise elution mode with different proportions of methylene dichloride and methanol to give yield of about 72% and purity of >95%. The maceration method developed by Malawade et. al. used methylene dichloride as a solvent for extraction followed by flash column chromatography on silica with elution by step gradient mode using hexane and ethyl acetate mixture. In this method the crystallisation was performed by antisolvent addition followed by cooling to give about 37% overall yield of the process and purity of >99% for artemisinin. The Liu et al developed a purification method from an ethanol extract by using a diatomite based purification protocol. This purification method uses cooling crystallisation followed by antisolvent re- crystallisation using hot methanol and water to give overall yield of 60% with >98% purity of artemisinin. The computationally designed adsorbents were used by Piletska et al to purify artemisinin as an alternative to silica based purification processes. This method uses two step antisolvent crystallisation processes to give overall yield of approximately 87% with purity of about 75%. The Qu et al developed a chromatography-crystallisation hybrid process which involves two step anti-solvent crystallisation process in which first step gives yield of 30% with 95% purity for artemisinin whereas second step gives the yield of 50% with purity of 73% for artemisinin.

Most of the methods reported above for the purification gave artemisinin with purity of >98% and yield varying from 38% to 87%. This indicates that the recovery of highly pure artemisinin is difficult. The reduced yield may be due to the more number of steps inyolved in the process from extraction to the crystallisation. Also this loss in yield may be due to the presence of structurally similar co-metabolites and solubility enhancers in the waxy fractions of the extract. The economic viability of any process depends on the productivity of the process. There is a need for the development of the process for artemisinin purification which should give high recovery and high purity to make it economically viable. This can be achieved by reducing the number of steps in the process and removal of the structurally similar metabolites and solubility enhancers before the crystallisation step.

Object of the invention

The main object of the present invention is to provide a simple and robust process for extraction and preparative scale chromatographic purification of artemisinin.

Further object of the present invention is to develop a novel hybrid process for purification of artemisinin in high yield and high purity by the removal of structurally similar co-metabolites and the solubility enhancers.

The conventional processes for artemisinin extraction involves solvent extraction followed by liquid-liquid extraction, crystallisation/re-crystallisation and repeated chromatographic steps to obtain artemisinin.

Further object of the present invention relates to the process for the extraction and purification of artemisinin using normal phase affinity chromatography and bleaching. The process has the advantage of using minimum number of solvents, minimum number of steps, high productivity and use of reusable polymeric adsorbents/matrix leading to better techno- economic viability.

A very important object of the invention is that the conventional processes have used the silica column chromatography whose reusability is less compared to polymeric adsorbents/matrix. The conventional processes use 4 to 5 solvents for the entire process involving antisolvent crystallisation and re-crystallisation which decreases the techno- economic viability of the process.

Summary of invention

The present invention provides a simple and robust, novel hybrid process for the extraction and chromatographic purification of artemisinin with high productivity and high purity. In the process of the present invention the organic solvent extract was treated with ion exchange matrix in negative mode of interaction. This was followed by the bleaching of the outlet stream of the extract. The bleached extract was subjected to the normal phase affinity chromatography and then selectively eluted by using mixture of organic solvents. The elution fractions obtained from the normal phase affinity chromatography were subjected to bleaching followed by evaporative crystallisation to obtain artemisinin in crystalline white solid form. It is the first report and new invention over the prior art that the ion exchange chromatography and normal phase affinity chromatography are coupled with bleaching to remove the impurities and recover the artemisinin in high productivity and high purity.

Detailed description of the invention

The present invention provides a process for purification of artemisinin to get with yield of >90% and HPLC assay purity of >99%, which involves the steps of chromatographic purification coupled with bleaching to give artemisinin in high yield and high purity.

In an embodiment of the present invention, with mixture of organic solvent followed by filtration to remove the insoluble material from the extract. The filtrate/extract thus obtained was passed through ion exchange matrix for partial purification/decolorisation to remove colored impurities/pigments. The flow through fraction coming out from the ion exchange column was bleached to remove the colored impurities followed by the normal phase affinity chromatography. This was followed by elution using mixture of organic solvents to elute the bound product from affinity matrix. This elution fraction thus obtained was bleached to remove the impurities/solubility enhancers followed by evaporative crystallisation under reduced pressure to obtain pure crystals of artemisinin.

The invention relates to the process for the extraction and purification of artemisinin using normal phase affinity chromatography and bleaching. The process has the advantage of using minimum number of solvents, minimum number of steps, high productivity and use of reusable polymeric adsorbents/matrix leading to better techno-economic viability. The present invention involves the use of only two solvents throughout the process involving solid-liquid extraction, chromatography, bleaching and crystallisation. Most of the conventional processes have used the silica column chromatography whose reusability is less compared to polymeric adsorbents/matrix. The conventional processes use 4 to 5 solvents for the entire process involving antisolvent crystallisation and re-crystallisation which decreases the techno-economic viability of the process. The present provisional disclosure of the process of the present invention can be carried out in following steps as follows:

Steps:

1. Extraction of artemisinin using techniques such as but not limited to maceration, percolation, soaking, stirred extraction, packed column extraction, microwave assisted extraction or ultrasound assisted extraction with non polar or mixture of non polar and intermediate polar organic solvent system such as hexane, petroleum ether, ethyl acetate, methylene dichloride, ethyl formate etc. and a period in the range of 10 min to 24 hrs at temperature in the range of 25 to 45 °C.

2. Solid-liquid separation using filtration or decantation to get the extract or miscella (filtrate)

3. Concentration of the extract obtained in step 2 using evaporation/distillation of the extract under reduced pressure to obtain the concentrated/dry/semisolid crude extract OR

4. Alternatively the extract or miscella obtained in the step 2 can be used as it is for next step.

5. Contacting the extract or miscella obtained in step 3 or 4 with ion exchange adsorbent to remove the colored impurities. The flow through coming out from the ion exchange column is collected.

6. The flow through fractions obtained in step 5 are refined using bleacher (organic or inorganic material) such as but not limited to rice husk, saw dust, corn cob, barley husk, activated charcoal, diatomaceous earth, date pits, wheat straw, alumina, clay, silica beads, zeolites etc.

7. The step 6 is followed by filtration or decantation to separate bleacher from the refined miscella.

8. The refined miscella thus obtained in step 7 is concentrated by distillation or evaporation under reduced pressure to obtain the concentrated miscella.

9. The concentrated miscella obtained in step 8 is contacted with normal phase affinity media/adsorbent/matrix. The flow through fractions were collected and distilled or evaporated under reduced pressure to obtain unbound products/impurities.

10. The bound product in the step 9 is selectively eluted using mixture of non polar and intermediate polar organic solvent. The solvents used include such as but not limited to hexane, petroleum ether, ethyl acetate, methylene dichloride etc. The composition of solvent varies from 0.1% of intermediate polar solvent in non polar solvent to 100% of intermediate polar solvent.

11. The elution fractions obtained in the step 10, optionally bleached and filtered, and were concentrated by evaporation or distillation under reduced pressure to obtain the pure crystalline product of artemisinin.

In one of the embodiment of the process of the present invention the extraction is done by using maceration, percolation, soaking, stirred extraction, packed column extraction, microwave assisted extraction or ultrasound assisted extraction or combination thereof using non polar or mixture of non polar and intermediate polar organic solvent system such as hexane, petroleum ether, ethyl acetate, methylene dichloride, ethyl formate.

In the preferred embodiments of the process of the present invention wherein a process for the extraction of artemisinin from dried Artemisia annua leaves uses solvent in combination of non polar solvent such as hexane and intermediate polar solvent selected from but not limited to ethyl acetate, ethyl formate, methylene dichloride.

Yet another embodiment of the process of the present invention discloses that solvent extract or miscella obtained is treated with adsorbent matrix where partial purification of artemisinin takes place in negative mode. The separation takes place because of interaction of colour impurities with adsorbent matrix. The embodiment also discloses that the adsorbent used for partial purification is ion exchange matrix behaving as negative normal phase chromatography.

In the other embodiment of the process of the present invention that discloses the refining of the miscella obtained from adsorbent matrix is done by bleaching. The bleaching involves the use of material such as but not limited to organic material such as rice bran, wheat straw, saw dust, activated charcoal or inorganic material such silica, alumina, diatomaceous earth etc.

Yet another embodiment of the process of the present invention discloses that the refined miscella is contacted with the normal phase affinity matrix or adsorbent where artemisinin binds more selectively than the impurities and is then selectively eluted using solvent combination of non polar and intermediate polar sovents selected from but not limited to hexane, ethyl acetate, ethyl formate, methylene dichloride etc.

In the embodiment of the process of present invention the adsorbent matrix used in column chromatography comprises of one or more of following (i) a non sulfonic resin (ii) non ionic resin (iii) an anion exchange resin (iv) having a surface and/or surface group, which has interacting ability with impurities (v) which is rigid and porous, (vi) in the form of a membrane, (vii) has synthetic or natural polymeric base matrix, (viii) has a synthetic base matrix of polystyrene-divinylbenzene (PSDVB), polymethacrylates, polyacrylamide and the like, (ix) has natural polymeric base matrix of agarose, cellulose, chitosan, dextran and the like, (x) is crosslinked, (xi) a modified silica with aromatic and/or aliphatic moiety as substituted group having CI to C30 carbon atoms, (xii) has interacting group which is a part of base matrix or grafted on the base matrix by known activation chemistry, (xiii) the said interacting group is unsaturated or saturated aliphatic and/or an aromatic moiety of a C1-C30 carbon molecules, (xiv) has the interacting group which is halogen atom, (xv) the interacting group is cyano, diol or amino, (xvi) has the interacting group which has different interacting ability for artemisinin, colouring matter and other structural impurities related to artemisinin and solubility enhancers present in the waxy material (xvii) microporous, macroporous, mesoporous, gigaporous and super-macroporous (xviii) a mixed mode or anion exchange matrix based on one or more than one of a synthetic or natural polymeric matrix and having amino (primary, secondary or tertiary) or imino moiety, (xix) a matrix based on one or more of a polymer comprising PSDVB, polymethacrylates, polyacrylamide, a natural polymer and combinations thereof having hydroxyl or diol group, (xx) a hydrophobic group.

In one of the embodiment of the present invention of the process for purification of artemisinin the adsorbent used for partial purification or decolorisation is one or more of the following but not limited to Diaion HPA25, Diaion HPA75, Indion 830ss, Indion 810, Indion 890, Indion 830, Indion 860, Diaion PA308, Diaion PA312, Diaion PA316, Diaion WA20, Diaion WA21J, Diaion WA30, Unosphere Q. The interacting group of the adsorbent matrix may be the part of base matrix, or may be grafted on the matrix before or after its preparation by the known activation chemistry, to give hydrophobic, hydrophilic, polar and/or ionic characteristics to the adsorbent matrix or resin.

In another embodiment of the process of present invention the artemisinin is obtained in high purity and high yield from crude extract obtained from dried leaves of Artemisia annua involves the chromatographic purification process wherein the process is carried out in one or more of a mode comprising in single or in multiples or a combination of a batch mode, a continuous mode, an expanded bed, a fluidized bed, a liquid solid circulating fluidized bed (LSCFB), a simulated moving bed (SMB), a moving bed, an improved simulated moving bed (ISMB), a centrifugal chromatography, an annular chromatography; membrane chromatography with adsorption being preferably performed with a packed bed chromatographic column or expanded bed chromatographic column, which comprises packing the column with a suitable adsorbent and passing the solution of crude extract or solvent extract and mobile phase/s through the column.

Yet another embodiment of the process of the present invention discloses that the adsorbent used for chromatography are regenerated for use in next cycle using organic solvents including but not limited to ethyl acetate, ethyl formate, methylene dichloride, ethylene dichloride, methyl formate etc.

Another embodiment of the process of the present invention discloses that the chromatography columns are operated from 10 cm/hr to 1000 cm/hr linear velocity for all the steps including equilibration, feed loading, washing, elution and regeneration.

In one of the embodiment of the process of present invention for purification of artemisinin, the bleacher used in refining step for elution fractions from normal phase affinity matrix may be identical or different. The use of type of bleacher is decided according to the type and level of impurities present in the elution fractions.

The other embodiment of the process of the present invention discloses that the final solid product is obtained by crystallisation more preferably by evaporation under reduced pressure.

The other embodiment of the process of the present invention discloses that the chromatography columns are operated at temperature ranging between 25 to 45 °C.

Example 1:

The lOOg dried leaves of Artemisia containing 0.5% of artemisinin were extracted with three volumes of 400 ml intermediate polar solvent in hexane. The extract obtained in each step is pooled and filtered to remove the particulate matter. The filtered extract or miscella was loaded onto the first chromatography column filled with ion exchange matrix at linear velocity of lOOcm/hr. The flow through fractions from the first chromatography column were collected and refined with bleacher at temperature not exceeding 50°C for 30min. The refined miscella was concentrated 8-1 OX and loaded onto the second chromatography column filled with the normal phase affinity media. The flow through fractions containing terpene oils were separately collected and wash with 3-4 bed volumes of non polar solvent was given to collect the terpene oil remained onto the column. The artemisinin was selectively eluted at a linear velocity of lOOcm/hr with 6-8 bed volumes mixture of intermediate polar solvent in hexane and refined with bleacher to remove the structurally related impurities and solubility enhancers present in elution fractions. The bleached elution fractions then concentrated 8- 10X by evaporation under reduced pressure to obtain the 0.45g (i.e. 90% yield based on content in the plant) crystalline artemisinin with HPLC assay purity of >99%.

Claims

We Claim,

1. A novel process for purification of artemisinin to get high yield of >90% and high purity of >99%. by chromatographic purification method coupled with bleaching, comprising the following steps,

a) extraction mixture of organic solvent followed by filtration to remove the insoluble material from the extract, which passed through ion exchange matrix(column) for partial purification/decolourisation to remove colored impurities/pigments.

b) The flow through fraction coming out from the ion exchange column was bleached, followed by the normal phase affinity chromatography,

c) elution of mixture obtained at step (b) using mixture of organic solvents to elute the bound product from affinity matrix, agaisn bleached to remove the impurities/solubility enhancers followed by evaporative crystallisation under reduced pressure to obtain pure crystals of artemisinin.

2. A process as claimed in claims 1, wherein the use of only two solvents throughout the process involving solid-liquid extraction, chromatography, bleaching and crystallisation,

3. A process as claimed in above claims wherein the extraction, microwave assisted extraction or ultrasound assisted extraction with non polar or mixture of non polar and intermediate polar organic solvent system such as hexane, petroleum ether, ethyl acetate, methylene dichloride, ethyl formate at a period in the range of 10 min to 24 hrs at temperature in the range of 25 to 45 °C.

4. A process as claimed in claim 1, wherein the solid-liquid separation using filtration or decantation to get the extract or miscella (filtrate),

5. A process as claimed in claim 4, wherein the crude extract contacting the extract or miscella with ion exchange adsorbent to remove the colored impurities, and collected the flow coming out from the ion exchange column, which allowed to refined using bleacher (organic or inorganic material) rice husk, saw dust, corn cob, barley husk, activated charcoal, diatomaceous earth, date pits, wheat straw, alumina, clay, silica beads, zeolites followed by decantation to separate bleacher from the refined miscella.

6. A process as claimed in claim 5 wherein the refined miscella is concentrated by distillation or evaporation under reduced pressure to obtain the concentrated miscella.

7. A process as claimed in claim 6 wherein the concentrated miscella obtained is contacted with normal phase affinity media/adsorbent/matrix, the flow through fractions were collected and distilled or evaporated under reduced pressure to obtain unbound products/impurities.

8. A process as claimed in claim 7 wherein the product obtained is selectively eluted using mixture of non polar and intermediate polar organic solvent, The solvents used are selected from hexane, petroleum ether, ethyl acetate, methylene dichloride etc. the mxture of solvent varies from 0.1% of intermediate polar solvent in non polar solvent to 100% of intermediate polar solvent, optionally bleached and filtered to obtain refined miscella as in claim 5, and followed by concentration by evaporation or distillation under reduced pressure to obtain the pure crystalline product of artemisinin.

9. A process as claimed in claim 8 wherein the mixture solvent are taken from the or combination thereof using non polar or mixture of non polar and intermediate polar organic solvent system such as hexane, petroleum ether, ethyl acetate, methylene dichloride, ethyl formate.

10. A process as claimed in the above claims wherein the bleaching involves the use of material such as organic material such as rice bran, wheat straw, saw dust, activated charcoal or inorganic material such silica, alumina, diatomaceous earth etc., and adsrobents are (i) a non sulfonic resin (ii) non ionic resin (iii) an anion exchange resin (iv) having a surface and/or surface group, which has interacting ability with impurities (v) which is rigid and porous, (vi) in the form of a membrane, (vii) has synthetic or natural polymeric base matrix, (viii) has a synthetic base matrix of polystyrene-divinylbenzene (PSDVB), polymethacrylates, polyacrylamide and the like, (ix) has natural polymeric base matrix of agarose, cellulose, chitosan, dextran and the like, (x) is crosslinked, (xi) a modified silica with aromatic and/or aliphatic moiety as substituted group having CI to C30 carbon atoms, (xii) has interacting group which is a part of base matrix or grafted on the base matrix by known activation chemistry, (xiii) the said interacting group is unsaturated or saturated aliphatic and/or an aromatic moiety of a C1-C30 carbon molecules, (xiv) has the interacting group which is halogen atom, (xv) the interacting group is cyano, diol or amino, (xvi) has the interacting group which has different interacting ability for artemisinin, colouring matter and other structural impurities related to artemisinin and solubility enhancers present in the waxy material (xvii) microporous, macroporous, mesoporous, gigaporous and super-macroporous (xviii) a mixed mode or anion exchange matrix based on one or more than one of a synthetic or natural polymeric matrix and having amino (primary, secondary or tertiary) or imino moiety, (xix) a matrix based on one or more of a polymer comprising PSDVB, polymethacrylates, polyacrylamide, a natural polymer and combinations thereof having hydroxyl or diol group, (xx) a hydrophobic group.